Primary Structure of Proteins Sequence Analysis by Tandem Mass Spectrometry

Analysis of the mixture using matrix-assisted desorption ionization mass spectrometry (section 5.3) allows for direct sequence determination from the snccessive mass differences of the peptide ladder. The application of the volatile trifluoroethyl isothiocyanate results in a significant optimization of this procedure and allows for peptide sequencing at the femtomole level (Bartlet-Jones et al., 1994). C-terminal ladder sequencing uses ammonium thiocyanate in acetic anhydride coupled with mass spectrometric analysis of truncated peptides (Thiede et al, 1997). Matrix-assisted desorption ionization instruments with delayed extraction (Brown and Leimon, 1995) allow for the discrimination of aU amino acids, except Leu and He. 

3 PRIMARY STRUCTURE OF PROTEINS SEQUENCE ANALYSIS BY TANDEM MASS SPECTROMETRY 

TABLE 5.2 Experimental conditions for electrospray, micospray and nanospray ionization 

Approx, limit of detection 10 femtomole 1 femtomole 50 attomole 

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Edman degradation was originally developed for determination of the primary structure (i.e., amino acid sequence) of peptides and proteins. Sequence analysis is not regularly performed for quality control in routine peptide synthesis but is more often employed for problem solving. As described earlier in this chapter, efficient characterization of synthetic peptides can be readily obtained by a combination of RP-EDPLC and mass spectrometry. Amino acid analysis is also valuable if MS is not available. If an incorrect mass or a discrepancy in the amino acid composition is found, one obvious alternative is to resynthesize the peptide. But, in order to deduce the cause of a failed synthesis, additional analyses must be performed. Both Edman degradation and tandem MS can be used to obtain sequence information... 

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